Transport of organelles and macromolecular complexes through the cytoplasm is essential for every eukaryotic cell. This process is performed by motor proteins that use the chemical energy of ATP hydrolysis to move their cargo along microtubules and actin filaments. The spatial and temporal control of motor- dependent transport is critical for cell division, organelle transport and positioning, and the movement of mRNA and protein complexes within the cell. Defects in organelle transport and motor-associated proteins contribute or cause many neurodegenerative diseases, neurofibromatosis and defects of pigmentation. The goal of this proposal is to understand how multiple motors moving a cargo function together to achieve targeted and timely delivery of components in the cell. We want to know how the activity of multiple motors is coordinated in a cell and how these motors are regulated. Two biological models will be used for the proposed work. A permanent cell line of pigment cells (melanophores) from the frog Xenopus laevis will be used to study the regulation of movement. This is an ideal system for the analysis of regulation because the motors are well-characterized and movement of pigment organelles can be triggered by changes in cAMP concentration. Cultured neuronal and phagocytic cells from Drosophila melanogaster are extremely sensitive to protein knock-down by RNAi allowing for efficient functional analysis of components involved in motility and coordination of motors. This proposal has four specific aims: (i) to find the mechanisms of cross-talk between microtubule motors of the opposite polarity;(ii) to find if multiple motors of the same polarity make transport more efficient and determine to what extent movement of the microtubules contributes to cargo transport;(iii) to find how myosin motors, moving along a network of actin filaments, and intermediate filaments interacting with cargo, modulate long-range transport by microtubule motors;(iv) to find mechanisms of targeting signaling molecules to cargo organelles and the identity of components downstream of signaling molecules.